Cells grown on poly-d-lysine–coated glass-bottomed plates (MatTek Corporation) were transfected with desired constructs using Lipofectamine 2000. After 48 hours, cells were fixed in 4% (w/v) paraformaldehyde and permeabilized in phosphate-buffered saline (PBS) containing 0.2% (v/v) Triton X-100 for 10 min at room temperature before staining with the appropriate antibodies diluted in PBS containing 3% (w/v) bovine serum albumin for 30 min. The primary antibodies used were GM130 (1:200) (BD Transduction Laboratories), IP3R (1:100) (Affinity BioReagents), and EEA1 (1:200) (Cell Signaling). Labeling by primary antibodies was followed by incubation in a 1:200 dilution of the relevant Alexa Fluor 546–conjugated secondary antibody (Invitrogen) for 30 min. All antibody incubation steps were followed by extensive washes in PBS. Laser-scanning confocal microscopy of fixed cells was performed with a Leica SP inverted confocal imaging microscope (Leica; Lasertechnik). Images were processed with ImageJ (National Institutes of Health) and Adobe Photoshop CS3.

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